1. Field of the Invention
The present invention provides a method for analyzing compounds with amino groups (e.g., amino acids, peptides, and amino acid analogues) rapidly and easily with higher sensitivity by liquid chromatography/mass spectrometry (LS/MS), as well as an apparatus therefor.
2. Discussion of the Background
Amino acids in biological organisms are useful for the discriminative determination of diseases, including diseases associated with abnormalities in the amino acid metabolism. Amino acids are used, for example, for the diagnosis of congenital aminoacidopathy, for the indicator of severity determination or therapeutic treatment of liver disfunction, and for the understanding of the conditions of patients at a state of malnutrition.
As the most famous example thereof, the synthesis of valine, leucine and isoleucine, referred to as branched-chain amino acids (BCAA), is reduced in severe liver diseases such as fulminant hepatitis and liver impairment. Since phenylalanine, an aromatic amino acid (AAA), is metabolized mainly in liver, the blood concentration thereof is increased when the metabolism of phenylalanine is inhibited. Consequently, the concentration ratio of BCAA to AAA is reduced to a smaller value, and the level reflects the severity of liver diseases. The ratio BCAA/AAA is generally called Fisher ratio and has been traditionally used for the determination of liver disease severity (see, for example, Fisher J E, et al., “Surgery”, 1976, July, Vol. 80, p. 77-91). Additionally, it is known that diabetes mellitus involves the increase of phenylalanine, tyrosine, isoleucine, leucine and valine and the decrease of alanine (see, for example, Felig P, et al., “Diabetes”, 1970, October, Vol. 19, p. 727-728).
With respect to methods for analyzing amino acids, amino acid analyzers based on post-column derivatization are frequently used. According to the methods, generally, amino acids are separated on a cation exchange column, followed by color reaction with ninhydrin. However, the methods have a drawback of long analytical time. Recently, the analytical time of a standard analytical method for analytes of about 20 kinds of amino acids as protein hydrolysates is approximately 20 minutes due to the modifications of separation columns, buffer flow and the like (see, for example, official gazette of JP-A-2002-243715). Meanwhile, methods for analyzing amino acids on the basis of pre-column derivatization reaction are significantly studied. Any amino acid composing protein can be analyzed in about 4.5 minutes by a pre-column derivatization reaction process using phenylisothiocyanate as a reagent. By the LC-MS method using an analytical reagent developed by the present inventors, additionally, 20 kinds of amino acids are possibly analyzed in 2.8 minutes, as described (see, for example, Pamphlet of International Publication WO 03/069328). However, leucine and isoleucine are not separated from each other in this example. Therefore, leucine and isoleucine cannot be individually analyzed simultaneously.
Not only amino acids that compose proteins, but also a great number of amino acid analogues exist in biological organisms. Typically, the amino acid analogues include, for example, taurine, O-phosphoethanol amine, hydroxyproline, methionine sulfoxide, sarcosine, α-aminoadipic acid, citrulline, α-amino-n-butyric acid, pipecolic acid, homocysteine, homocitrulline, alloisoleucine, saccharopine, cystathionine, argininosuccinic acid, cysteine-homocysteine, β-alanine, aminolevulinic acid, β-aminoisobutyric acid (β-AiBA), γ-amino-n-butyric acid (GABA), homocystine (Hcys2), argininosuccinate anhydride, hydroxylysine, aminoethylcysteine, ornithine, 1-methylhistidine, and 3-methylhistidine. Various physiological functions are going to be discovered or elucidated in these amino acid analogues contained in biological fluids.
According to an analytical method on the principle of post-column derivatization, comprising separating amino acids and amino acid analogues (these are collectively referred to as compounds with amino group) on a cation exchange column and subsequent color reaction with ninhydrin, the analytical time for these 41 components of compounds with amino groups is 60 minutes at minimum; and the analytical time for 53 components is 148 minutes at minimum. (See, for example, official gazette of JP-A-2002-71660). Such a long analytical time is a serious factor disturbing research in regard to the physiological functions of amino acids. Even when a novel discovery into the relationship between amino acids and health or diseases is found, the information cannot effectively be employed due to processing delays and deficiencies in processing ability.